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  • 94
    Thermo Fisher p ctp
    P Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p ctp - by Bioz Stars, 2020-08
    94/100 stars
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    90
    GE Healthcare p ctp
    P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer p ctp
    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM <t>PUGNAc</t> were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 <t>P-CTP</t> /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.
    P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    DuPont de Nemours p ctp
    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM <t>PUGNAc</t> were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 <t>P-CTP</t> /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.
    P Ctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ICN Biomedicals p ctp
    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM <t>PUGNAc</t> were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 <t>P-CTP</t> /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.
    P Ctp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    NEN Life Science p ctp
    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM <t>PUGNAc</t> were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 <t>P-CTP</t> /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.
    P Ctp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Valiant p ctp
    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM <t>PUGNAc</t> were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 <t>P-CTP</t> /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.
    P Ctp, supplied by Valiant, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare α32 p ctp
    <t>CTP</t> and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows
    α32 P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    PerkinElmer α32 p ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    α32 P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 89/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    DuPont de Nemours α32 p ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    α32 P Ctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ICN Biomedicals α 32 p ctp
    Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 <t>P]CTP-labeled</t> tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.
    α 32 P Ctp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant α 32 p ctp
    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 <t>P-CTP</t> and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
    α 32 P Ctp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science α 32 p ctp
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    α 32 P Ctp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    HARTMANN ANALYTIC α 32 p ctp
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    α 32 P Ctp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer mci α32 p ctp
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    Mci α32 P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Pharmaceuticals p ctp
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    P Ctp, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    PerkinElmer p ctp substrate
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    P Ctp Substrate, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    DuPont de Nemours a32 p ctp
    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 <t>P]CTP</t> as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.
    A32 P Ctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM PUGNAc were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 P-CTP /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.

    Journal: bioRxiv

    Article Title: O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System

    doi: 10.1101/2020.01.23.917237

    Figure Lengend Snippet: OGT is required to establish a paused pol II. A. OGT inhibitor addition after PIC formation causes release of paused pol II. 40 μM STO4 or 3mM PUGNAc were added simultaneously with CMV promoter DNA or 30’ afterwards (to allow PICs to form). These reactions were then assayed by standard pulse-chase assay. B. OGT inhibition causes release of paused pol II. The indicated final concentrations of either STO4 or STO6 were added to preformed PICs which were then assayed by pulse-chase. C. Three OGT inhibitors affect pause release. Either DMSO, 80 μM OSMI-2a or 160 μM OSMI-4a were added after PIC formation followed by a standard pulse-chase assay (lanes 1-4). Lanes 5-7 show a titration of STO4 (40, 80, 200, 400 μM final concentrations). D. Schematic shows the steps in the immobilized template assay in panel E. E. rOGT rescues the OGT inhibitor STO4 aberrant elongation effect. Immobilized templates were created by allowing PICs for form for 30’, followed by a 15’ incubation with 0.1 mM STO4 and then a 30’’ pulse with 32 P-CTP /GUA followed by addition of EDTA to stop transcription. Beads were washed as indicated to remove all rNTPs, STO4, and nuclear extract. Individual reactions were suspended in transcription buffer and either rOGT or rOGT and 0.4 mM UDP-GlcNAc were added as indicated, for 15’, followed by a 5’ chase with unlabeled NTPs.

    Article Snippet: Reagents ST045849 and ST060266 (TimTec), Flavopiridol (Sigma F3055), PJ34, THZ531 (APExBIO, A8736), PUGNAc (Sigma A7229), 32 P-CTP (3000 Ci/mmol, Perkin Elmer), NTPs (Roche), Sarkosyl (Sigma), WGA-agarose, GlcNAc (Sigma), UDP-GlcNAc (Sigma).

    Techniques: Pulse Chase, Inhibition, Titration, Incubation

    Super Elongation Complex (SEC) and P-TEFb stimulate release of paused RNA pol II A. Shown is a schematic of steps in the immobilized template assays in panels C and F. B. Purification of rPTEFb. Baculovirus expression vectors containing human P-TEFb and cyclin T1 were expressed in Sf9 cells and purified by Ni-Sepharose affinity chromatography. C. Pol II elongation is stimulated by rP-TEFb. Pulsed immobilized templates were isolated after they had been isolated and washed to remove 32 P-CTP/GUA and nuclear extract. The indicated factors (P.3c is a concentrated P11 0.3M elution that contains some elongation activity-see lane 2) were added to immobilized templates and incubated for 15’, followed by a chase with unlabeled rNTPs. rP-TEFb plus a P11 elongation fraction (P.3c) was added to immobilized templates and compared to either no additional factors (lane 1) or the P.3c fraction alone (lane 2) as indicated in panel A. D. Purification of F-SEC. Flag-tagged SEC subunit AFF1 was purified from 293 cells using M2-agarose and eluted by excess Flag peptide. Input extract contained undetectable levels of SEC subunits, but which were concentrated by affinity purification (E1-E3), as indicated by western blot analysis for SEC subunits AFF1, ELL2, and CDK9. E. Kinase activity of F-SEC was assessed by incubating purified F-SEC (lane 2) or rP-TEFb (positive control, lane 3) with the known substrate GST-CTD, containing the first 26 repeats of human pol II CTD. Phosphorylation was detected by western blot using an anti-phosphoserine 2 CTD antibody. Lane 1 contains only the GST-CTD and the kinase reaction buffer. F. Schematic shows the steps in the immobilized template assay in panel G. G. F-SEC stimulates release of paused pol II. Either purified F-SEC or the control elution buffer were titrated into pulsed immobilized templates after they had been isolated and washed to remove 32 P-CTP /GUA and nuclear extract. After a 15’ incubation, unlabeled NTPs were added for 5’ to chase any elongation competent pol II into making full-length RNAs.

    Journal: bioRxiv

    Article Title: O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System

    doi: 10.1101/2020.01.23.917237

    Figure Lengend Snippet: Super Elongation Complex (SEC) and P-TEFb stimulate release of paused RNA pol II A. Shown is a schematic of steps in the immobilized template assays in panels C and F. B. Purification of rPTEFb. Baculovirus expression vectors containing human P-TEFb and cyclin T1 were expressed in Sf9 cells and purified by Ni-Sepharose affinity chromatography. C. Pol II elongation is stimulated by rP-TEFb. Pulsed immobilized templates were isolated after they had been isolated and washed to remove 32 P-CTP/GUA and nuclear extract. The indicated factors (P.3c is a concentrated P11 0.3M elution that contains some elongation activity-see lane 2) were added to immobilized templates and incubated for 15’, followed by a chase with unlabeled rNTPs. rP-TEFb plus a P11 elongation fraction (P.3c) was added to immobilized templates and compared to either no additional factors (lane 1) or the P.3c fraction alone (lane 2) as indicated in panel A. D. Purification of F-SEC. Flag-tagged SEC subunit AFF1 was purified from 293 cells using M2-agarose and eluted by excess Flag peptide. Input extract contained undetectable levels of SEC subunits, but which were concentrated by affinity purification (E1-E3), as indicated by western blot analysis for SEC subunits AFF1, ELL2, and CDK9. E. Kinase activity of F-SEC was assessed by incubating purified F-SEC (lane 2) or rP-TEFb (positive control, lane 3) with the known substrate GST-CTD, containing the first 26 repeats of human pol II CTD. Phosphorylation was detected by western blot using an anti-phosphoserine 2 CTD antibody. Lane 1 contains only the GST-CTD and the kinase reaction buffer. F. Schematic shows the steps in the immobilized template assay in panel G. G. F-SEC stimulates release of paused pol II. Either purified F-SEC or the control elution buffer were titrated into pulsed immobilized templates after they had been isolated and washed to remove 32 P-CTP /GUA and nuclear extract. After a 15’ incubation, unlabeled NTPs were added for 5’ to chase any elongation competent pol II into making full-length RNAs.

    Article Snippet: Reagents ST045849 and ST060266 (TimTec), Flavopiridol (Sigma F3055), PJ34, THZ531 (APExBIO, A8736), PUGNAc (Sigma A7229), 32 P-CTP (3000 Ci/mmol, Perkin Elmer), NTPs (Roche), Sarkosyl (Sigma), WGA-agarose, GlcNAc (Sigma), UDP-GlcNAc (Sigma).

    Techniques: Purification, Expressing, Affinity Chromatography, Isolation, Activity Assay, Incubation, Affinity Purification, Western Blot, Positive Control

    CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Journal:

    Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

    doi: 10.1038/emboj.2008.124

    Figure Lengend Snippet: CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Article Snippet: The assay procedures for CMP incorporation into mini-D73 N74 were the same as those described above, except that 100 μM CTP, 100 μM ATP, and 100 nM α-32 P CTP (3000 Ci/mmol; GE Healthcare) were used instead of the 100 μM ATP and 100 nM α-32 P ATP.

    Techniques: Mutagenesis

    Detection of positive-sense RNA synthesis by DI-19, DI-19(D10), and SFV in BHK-21 cells by Northern blotting. SFV-infected cells were lipofectamine transfected with DI RNAs transcribed from pSFVDI-19 and pSFVDI-19(D10). Cells were infected with SFV at 0 h, transfected from 1 to 3 h postinfection, and sampled at 3 to 8 h postinfection. Cytoplasmic RNA was quantified by spectrophotometry, denatured with glyoxal, and electrophoresed on agarose gel. After blotting, RNA was probed with a [γ- 32 P]CTP-labelled RNA probe (N594-ve). Lanes: 1, DI-19(D10); 2, DI-19; 3, SFV; 4, noninfected, nontransfected cells. Two cell bands were present in all lanes, presumably as a result of nonspecific hybridization of the probe to rRNAs. Only the larger (upper arrow) is visible at this exposure.

    Journal: Journal of Virology

    Article Title: Deletion Analysis of a Defective Interfering Semliki Forest Virus RNA Genome Defines a Region in the nsP2 Sequence That Is Required for Efficient Packaging of the Genome into Virus Particles

    doi:

    Figure Lengend Snippet: Detection of positive-sense RNA synthesis by DI-19, DI-19(D10), and SFV in BHK-21 cells by Northern blotting. SFV-infected cells were lipofectamine transfected with DI RNAs transcribed from pSFVDI-19 and pSFVDI-19(D10). Cells were infected with SFV at 0 h, transfected from 1 to 3 h postinfection, and sampled at 3 to 8 h postinfection. Cytoplasmic RNA was quantified by spectrophotometry, denatured with glyoxal, and electrophoresed on agarose gel. After blotting, RNA was probed with a [γ- 32 P]CTP-labelled RNA probe (N594-ve). Lanes: 1, DI-19(D10); 2, DI-19; 3, SFV; 4, noninfected, nontransfected cells. Two cell bands were present in all lanes, presumably as a result of nonspecific hybridization of the probe to rRNAs. Only the larger (upper arrow) is visible at this exposure.

    Article Snippet: Linearization at the Spe I site in the multiple cloning site allowed [γ-32 P]CTP (Amersham International plc., Aylesbury, UK) negative-sense DI-19 RNA transcripts to be produced from the T7 promoter.

    Techniques: Northern Blot, Infection, Transfection, Spectrophotometry, Agarose Gel Electrophoresis, Hybridization

    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis *

    doi: 10.1074/jbc.M110.151316

    Figure Lengend Snippet: Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Article Snippet: GTP was added at different concentrations before or at the same time as 0.5 m m ATP, 3′-dUTP, and 10 or 100 μ m CTP with 4 μCi of [α-32 P]CTP (3000 Ci·mmol−1 , PerkinElmer Life Sciences).

    Techniques: Incubation, TCA Precipitation

    Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

    Journal: Nucleic Acids Research

    Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA

    doi: 10.1093/nar/gkl530

    Figure Lengend Snippet: Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

    Article Snippet: [α-32 P]UTP, [α-32 P]CTP, [α-32 P]ATP and [α-32 P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics.

    Techniques: Modification, Sequencing, Labeling, Thin Layer Chromatography, Mutagenesis, Incubation, Autoradiography, Migration

    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 P-CTP and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.

    Journal: PLoS ONE

    Article Title: Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

    doi: 10.1371/journal.pone.0025837

    Figure Lengend Snippet: The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 P-CTP and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.

    Article Snippet: RdRp assays RdRp assays were performed as 20 µL reactions containing 20 mM sodium glutamate (pH 8.2), 12.5 mM DTT, 4 mM MgCl2 , 1 mM MnCl2 , 0.5% Triton X-100, 0.2 mM GTP, 0.1 mM ATP and UTP, 250 nM [α-32 P] CTP (MP Biomedicals), 2 pmol PE46 and 1 pmol LE19p as templates .

    Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Labeling, Electrophoretic Mobility Shift Assay

    Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 P]CTP as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.

    Journal: Journal of Virology

    Article Title: Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA

    doi: 10.1128/JVI.76.14.6944-6956.2002

    Figure Lengend Snippet: Determination of the initiation sites of RNA synthesis on X RNA by HCV NS5B. (A) Secondary structure of X RNA. The major initiation sites of RNA synthesis are indicated by the arrow on stem I. (B) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA product of HCV NS5B using the X RNA template. The bar indicates the major RdRp products on the gel. The numbers on the left give the length of the 5′-labeled X RNA partially digested by RNase T 1 (lane 1). The numbers on the right give the lengths of the RNA synthesis products of NS5B. Lane 2, partial alkaline hydrolysis of the 5′-labeled X RNA; lane 3, template X RNA labeled internally with [α- 32 P]CTP as a marker; lanes 4 and 5, RdRp product on the X RNA template before (−) and after (+) the 3′ end was blocked.

    Article Snippet: The RdRp reactions were performed in a 15-μl volume with 20 mM HEPES (pH 8.0), 1.5 mM MnCl2 , 100 mM ammonium acetate, 1 mM DTT, 500 μM GTP, 250 μM (each) ATP and UTP, 5 μM CTP, 10 μCi of [α-32 P]CTP (NEN Life Science Products), 12 pmol of RNA template, 10 U of recombinant RNasin (Promega), and 1 to 3 μmol of NS5B.

    Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Marker

    RdRp reaction of the point mutants of X RNA with HCV NS5B. (A) Nucleotide sequences in stem I of the mutant RNAs. The characters in boldface represent the elements for rendering the bulge structure. The nucleotides in italics indicate the pyrimidine-rich region in stem I. (B) Products of RdRp reaction on wild-type X RNA and its point mutants on a 7 M urea-8% polyacrylamide gel. The length of the marker RNA is indicated at the left of the gel. Lane 1, template X RNA internally labeled with 32 P; lanes 2 to 7, RdRp products of X, Xmut (1), Xmut (2), Xmut (3), Xmut (4), and Xmut (5) RNAs, respectively. (C) An 8 M urea-5% polyacrylamide sequencing gel electrophoresis autoradiogram showing the de novo synthesis products of X RNA and the point mutant RNAs before (−) or after (+) treatment with NaIO 4 . The template RNAs for directing RNA synthesis by NS5B are indicated at the top of the gel. RNase T 1 , 5′-labeled X RNA partially digested by RNase T 1 ; OH, partial alkaline hydrolysis of the 5′-labeled X RNA; RNA, template X RNA labeled internally with [α- 32 P]CTP as a marker. The numbers on the left give the length of the RNA, and the bar at the left of the gel indicates the pyrimidine-rich region of X RNA.

    Journal: Journal of Virology

    Article Title: Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA

    doi: 10.1128/JVI.76.14.6944-6956.2002

    Figure Lengend Snippet: RdRp reaction of the point mutants of X RNA with HCV NS5B. (A) Nucleotide sequences in stem I of the mutant RNAs. The characters in boldface represent the elements for rendering the bulge structure. The nucleotides in italics indicate the pyrimidine-rich region in stem I. (B) Products of RdRp reaction on wild-type X RNA and its point mutants on a 7 M urea-8% polyacrylamide gel. The length of the marker RNA is indicated at the left of the gel. Lane 1, template X RNA internally labeled with 32 P; lanes 2 to 7, RdRp products of X, Xmut (1), Xmut (2), Xmut (3), Xmut (4), and Xmut (5) RNAs, respectively. (C) An 8 M urea-5% polyacrylamide sequencing gel electrophoresis autoradiogram showing the de novo synthesis products of X RNA and the point mutant RNAs before (−) or after (+) treatment with NaIO 4 . The template RNAs for directing RNA synthesis by NS5B are indicated at the top of the gel. RNase T 1 , 5′-labeled X RNA partially digested by RNase T 1 ; OH, partial alkaline hydrolysis of the 5′-labeled X RNA; RNA, template X RNA labeled internally with [α- 32 P]CTP as a marker. The numbers on the left give the length of the RNA, and the bar at the left of the gel indicates the pyrimidine-rich region of X RNA.

    Article Snippet: The RdRp reactions were performed in a 15-μl volume with 20 mM HEPES (pH 8.0), 1.5 mM MnCl2 , 100 mM ammonium acetate, 1 mM DTT, 500 μM GTP, 250 μM (each) ATP and UTP, 5 μM CTP, 10 μCi of [α-32 P]CTP (NEN Life Science Products), 12 pmol of RNA template, 10 U of recombinant RNasin (Promega), and 1 to 3 μmol of NS5B.

    Techniques: Mutagenesis, Marker, Labeling, Sequencing, Nucleic Acid Electrophoresis

    RdRp reaction on the deletion mutants of X RNA by HCV NS5B. (A) Sequences of stem I of the deletion mutant RNAs. The nucleotides in italics indicate the pyrimidine-rich region in stem I. (B) RdRp reaction products of wild-type X RNA and its deletion mutants on a 7 M urea-8% polyacrylamide gel. The length of the marker RNA is indicated at the left of the gel. Lane 1, template X RNA with internally incorporated 32 P; lanes 2 to 9, RdRp products of X, Xdel (1), Xdel (2), Xdel (3), Xdel (4), Xdel (5), Xdel (11), and Xdel (18) RNAs, respectively. (C) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA products of HCV NS5B using X RNA and the deletion mutant RNA templates before (−) or after (+) treatment with NaIO 4 . RNase T 1 , 5′-labeled X RNA partially digested by RNase T 1 ; OH, partial alkaline hydrolysis of 5′-labeled X RNA; RNA, template X RNA labeled internally with [α- 32 P]CTP as a marker. The numbers on the left give the length of the RNA, and the bar at the left of the gel indicates the pyrimidine-rich region of X RNA.

    Journal: Journal of Virology

    Article Title: Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA

    doi: 10.1128/JVI.76.14.6944-6956.2002

    Figure Lengend Snippet: RdRp reaction on the deletion mutants of X RNA by HCV NS5B. (A) Sequences of stem I of the deletion mutant RNAs. The nucleotides in italics indicate the pyrimidine-rich region in stem I. (B) RdRp reaction products of wild-type X RNA and its deletion mutants on a 7 M urea-8% polyacrylamide gel. The length of the marker RNA is indicated at the left of the gel. Lane 1, template X RNA with internally incorporated 32 P; lanes 2 to 9, RdRp products of X, Xdel (1), Xdel (2), Xdel (3), Xdel (4), Xdel (5), Xdel (11), and Xdel (18) RNAs, respectively. (C) An 8 M urea-5% polyacrylamide gel electrophoresis autoradiogram showing the RNA products of HCV NS5B using X RNA and the deletion mutant RNA templates before (−) or after (+) treatment with NaIO 4 . RNase T 1 , 5′-labeled X RNA partially digested by RNase T 1 ; OH, partial alkaline hydrolysis of 5′-labeled X RNA; RNA, template X RNA labeled internally with [α- 32 P]CTP as a marker. The numbers on the left give the length of the RNA, and the bar at the left of the gel indicates the pyrimidine-rich region of X RNA.

    Article Snippet: The RdRp reactions were performed in a 15-μl volume with 20 mM HEPES (pH 8.0), 1.5 mM MnCl2 , 100 mM ammonium acetate, 1 mM DTT, 500 μM GTP, 250 μM (each) ATP and UTP, 5 μM CTP, 10 μCi of [α-32 P]CTP (NEN Life Science Products), 12 pmol of RNA template, 10 U of recombinant RNasin (Promega), and 1 to 3 μmol of NS5B.

    Techniques: Mutagenesis, Marker, Polyacrylamide Gel Electrophoresis, Labeling